In recent years, the use of the avidin-biotin complex has become well-known as am extremely versatile system, useful in a wide variety of bioanalytical applications including affinity chromatography, affinity cytochemistry, cell cytometry, blotting technology, diagnostics including immunoassays arid gene probe assays, hybridoma technology, bioaffinity sensors, drug delivery, and crosslinking, immobilization technology [Wilchek et al., Analytical Biochemistry 171:1-31 (1988) ].
The usefulness of the biotin-avidin system is characterized in terms of the high affinity (10.sup.15 M.sup.-1) of the biotin-avidin interaction, which accounts for the exceptional stability of this non-covalent interaction. Free biotin, or biotin in derivatized form (containing any of a number of reporter groups including fluorescent, radioisotope, electron-dense marker, enzyme, or immobilizing matrices) or biotin coupled to either low or high molecular weight molecules, is still recognized by avidin or streptavidin.
There have been several well-known applications of the biotin-avidin system including its use in enzyme-labeled immunoassays. In all of these the detection signal is achieved either by using a reporter-labeled avidin or streptavidin or by using native avidin or streptavidin and a biotinylated reporter.
U.S. Pat. No. 4,298,686, issued to Parikh et al. on Nov. 3, 1981, discloses a quantitative method for the determination of biological substances. In particular, an enzyme immunoassay is disclosed which utilizes a soluble biotin-tagged complex, in which biotin is covalently attached to an antibody molecule and a separation process is performed using avidin, which is immobilized on a solid phase.
U.S. Pat. No. 4,228,237, issued to Hevey et al. on Oct. 14, 1980, discloses the use of a biotin-avidin system for the determination of a ligand of interest in non-competitive and competitive binding processes. In particular, Hevey et al. disclose a method for the determination of a ligand of interest which utilizes enzyme-labeled avidin and biotin-labeled reagent in a specific binding process wherein the ligand to be determined is contacted with an insoluble phase containing a specific binding substance for the ligand. Following the specific binding reaction, the enzyme activity of either the insoluble phase or the liquid phase is assayed and thereby related to the amount of ligand in the medium.
U.S. Pat. No. 4,271,140, issued to Bunting on Jun. 2, 1981, discloses the use of noncovalent binding systems in double receptor specific binding assays in which a first receptor is bound by a second receptor, with the first receptor being capable of reversibly binding either a ligand or another receptor.
U.S. Pat. No. 4,659,678, issued to Forrest et al. on Apr. 21, 1987 discloses an immunoassay for detection of an antigen in a liquid sample wherein a complex is formed between antigen contained in the sample and two or more antibody reagents, the complex is bound to a solid support by non-covalent bonding, so that the amount of complex becoming bound to the support is determined and the process employs at least one monoclonal antibody. The biotin-avidin system is described as an example of a high affinity bonding system useful in the invention.
There are a number of limitations of the avidin-biotin system in diagnostic assays. One limitation is due to the occurrence of nonspecific binding, generally attributed to the basicity of the reporter-labeled avidin or streptavidin. Typically, non-specifically bound components cannot be washed away from the solid support systems, which can include polystyrene, polyacrylamide, nylon, crosslinked dextran of nitrocellulose paper, glass beads, plastic tubes or microtitre plates. The resulting signal noise is especially troublesome when the component to be assayed is present in very low concentrations. Usually, streptavidin is used instead of avidin in order to minimize non-specific binding.
Another limitation of an avidin-biotin system is that its use is generally restricted to measurement of the analyte of interest while it is bound to a solid support. In those cases where the reporter is an enzyme, practical difficulties associated with measurement while the complex remains bound to the solid phase can occur. The advantage of measuring an enzyme-labeled immuno-complex in free solution is that it reacts much more rapidly with a substrate in free solution than when bound to a solid support because reaction in free solution is less diffusion-limited and less sterically hindered. This circumstance provides a great advantage when immunoassays are carried out in automated diagnostic systems. Further, immunoassays requiring measurement of an analytically detectable group bound to a solid phase may be limited in that they require the use of special types of solid supports, such as those that are colorless and transparent.
The problems associated with measuring a reporter-labeled immuno-complex bound to a solid support, as described in the preceding paragraph, have stimulated efforts directed at releasing the labeled immuno-complex from the support into free solution. One approach is to so devise the chemistry that the immuno-complex is attached to the solid support through a chemical structure that can be cleaved by a chemical reaction such as reduction, oxidation and hydrolysis. For example, U.S. Pat. 4,231,999, issued to Carlsson et al. on Nov. 4, 1980, discloses a reagent for use with an immunochemical assay which entails an improved conjugating technique based on thiol-disulfide exchange. The reagent is characterized by the fact that it comprises a soluble conjugate of one or more molecules of immunoglobulin and one or more units of an analytically detectable group, in which molecules and units are bound together via bridges containing the cleavable --S--S-- group.
The use of chemically clearable bridges to attach immuno-complexes and the like to solid supports is elegant in concept but has proved to be of value only in special cases because the harsh conditions used to break the bridge may also cause damage elsewhere and thus reduce or destroy bioactivity required in the complex that is released. For this reason, the use of some non-covalent system involving a ligand and its binding partner as a bridge for joining components of an assay system, and a means of rapidly and gently displacing the ligand from its binding partner in order to subsequently separate the components of the assay system would De a more widely applicable technique.
Most applications of the biotin-avidin systems previously described are concerned only with tight and efficient binding. However, Hofmann et al. [Biochemistry 21: 978-984 (1982)] disclose that biotinyl-insulin and some of its analogs can bind non-covalently to succinoylavidin immobilized on AH Sepharose and can be released biospecifically by exposure of the loaded resin to buffers containing an excess of biotin. It may be noted here that succinoylavidin is a chemically modified avidin that has a lower isoelectric point than does avidin itself. It is thus less likely to cause problems with non-specific binding.
Lichstein et al. [Biochemical and Biophysical Research Communications 20(1): 41-45 (1965)] disclose the use of avidin and streptavidin for the characterization of analogs of biotin. Streptavidin, unlike avidin, was determined to be unable to bind the biotin analog dethiobiotin.
Finn et al. [Biochemistry, 23:2554-2558, (1984)] disclose a series of biotinylated and dethiobiotinylated insulins capable of forming complexes with succinoylavidins, in which the distance between the biotin carboxyl group and the insulin varies from 7-20 atoms. In contrast, Finn et al. further disclose the failure of dethiobiotin and its amide to bind to streptavidin, confirming the finding by Lichstein et al.
Ikariyama et al. [Analytical Chem. Symp. Series, 17 (Chem. Sensors): 693-698 (1983)] disclose a bioaffinity sensor for biotin which utilizes a membrane-bound azo dye-enzyme labeled avidin complex. Upon exposure to a solution containing biotin, the enzyme-labeled avidin is released from the membrane to form a stable avidin-biotin complex in solution.
There is a need for a specific binding assay which utilizes a releasable ligand, in a reversible binding displacement system in which a releasable ligand is rapidly displaced by a displacer ligand such that part of an immobilized complex which includes a reporter or an analytically detectable group or, at a minimum, the reporter or analytically detectable group itself, is released into a free liquid medium, and subsequently quantitatively measured. Further, there is a need for a specific binding assay which reduces or eliminates interference that may arise from non-specific binding of the reporter component to an insoluble phase.